Jeff Kramer jeff at COMPBIO.MED.WAYNE.EDU
Tue Aug 1 16:57:44 EST 1995

Mike Holloway wrote:
I'm giving up on the pET system and wondered if anyone else has had 
similar experiences.
	...stuff deleted...
Anyone had experience with pET vectors?

I have spentf years of my life trying to get lipoxygenase expression using
pET 3a while a grad student working on my PhD.  Even after I got expression, I
rarely got anywhere near reproducible amounts of protein.  The specific activity
was, thankfully, pretty reproducible.  By "pretty" I mean with in ~15%,
which was less than I would have liked, but good enough for my purposes.  What
it meant was that I did more repeat experiments than I probably would have had
to otherwise, to make sure my data was statistically significant.  Again, I was
fortunate in that three of the four site directed mutants that I made were dead
as door nails, and the forth hovered right around 50%, so even with the varia-
tion in specific activity, I could still say some meaningful things about lipoxy
genase.  Still, I wish I had chosen a different system.  I've even humbly sub-
mitted to my former boss that getting lox expression from yeast would make for
a nice Masters project for someone, and wouls save the entire lab a heck of a
lot of headache.  So in short, my advice is, give up on the pET vectors.  I
believe NEB now sells them, and I love NEB (no affiliation, etc).  I have never
gotten anything from them that wasn't excellant (I got the pET 3a before NEB
liscenced the system), but I wouldn't encourage anyone to use pET vectors, if
another system is readily available...


					Jeffrey A. Kramer, Ph.D.
					Wayne State University Medical Center
					C.S. Mott Center for Human Growth and
					275 E. Hancock
					Detroit, MI  48201
					jeff at compbio.med.wayne.edu

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